Cyclooxygenase inhibitors retard murine mammary tumor progression by reducing tumor cell migration, invasiveness and angiogenesis

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Abstract
Tumor‐derived prostaglandins (PGs) have been implicated in the progression of murine and human breast cancer. Chronic treatment with a non‐selective PG inhibitor indomethacin was shown in this laboratory to retard the development and metastasis of spontaneous mammary tumors in C3H/HeJ female retired breeder mice. The present study examined the role of endogenous prostaglandins in the proliferation/survival, the migratory and invasive behavior and angiogenic ability of a highly metastatic murine mammary tumor cell line, C3L5, originally derived from a C3H/HeJ spontaneous mammary tumor. This cell line was shown to express high levels of cyclooxygenase (COX) –2 mRNA and protein as detected by Northern and Western blotting as well as immunostaining. PGE2 production by C3L5 cells was primarily owing to COX‐2, since this was blocked similarly with non‐selective COX inhibitor indomethacin and selective COX‐2 inhibitor NS‐398, but unaffected with the selective COX‐1 inhibitor valeryl salicylate. C3L5 cell proliferation/survival in vitro was not influenced by PGs, since their cellularity remained unaffected in the presence of PGE2 or NS‐398 or PG‐receptor (EP1/EP2) antagonist AH6809; a marginal decline was noted only at high doses of indomethacin, which was not abrogated by addition of exogenous PGE2. Migratory and invasive abilities of C3L5 cells, as quantitated with in vitro transwell migration/invasion assays, were inhibited with indomethacin or NS‐398 or AH6809 in a dose‐dependent manner; the indomethacin and NS‐398‐mediated inhibition was partially reversed upon addition of exogenous PGE2. An in vivo angiogenesis assay that used subcutaneous implants of growth factor‐reduced matrigel inclusive of tumor cells showed a significant inhibition of blood vessel formation in these implants in animals treated with indomethacin compared with animals receiving vehicle alone. These studies show that selective and nonselective COX‐2 inhibitors retarded tumor progression in this COX‐2‐expressing murine mammary tumor model by inhibiting tumor cell migration, invasiveness and tumor‐induced angiogenesis. The inhibitory effects were not entirely PG dependent; some PG‐independent effects were also noted.